View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Am. J. Hum. Genet. 63:347–359, 1998 Identification of PEX10, the Gene Defective in Complementation Group 7 of the Peroxisome-Biogenesis Disorders Daniel S. Warren,1 James C. Morrell,1 Hugo W. Moser,2 David Valle,3 and Stephen J. Gould1,4 1Department of Biological Chemistry, 2The Kennedy Krieger Institute and Department of Neurology, 3The Howard Hughes Medical Institute and Departments of Pediatrics and Molecular Biology and Genetics, and 4Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore Summary Introduction The peroxisome-biogenesis disorders (PBDs) are a group Peroxisomes, which are present in almost all eukaryotic of genetically heterogeneous, lethal diseases that are cells, are single-membrane–bound organelles that par- characterized by neuronal, hepatic, and renal abnor- ticipate in numerous metabolic processes (Lazarow and malities; severe mental retardation; and, in their most Fujiki 1985). In humans, these processes include b-ox- severe form, death within the 1st year of life. Cells from idation of fatty acids; synthesis of bile acids, cholesterol, all PBD patients exhibit decreased import of one or more and plasmalogens; and a variety of H2O2-producing ox- classes of peroxisome matrix proteins, a phenotype idation reactions (van den Bosch et al. 1992). The en- shared by yeast pex mutants. We identified the human zymes responsible for these functions are encoded by orthologue of yeast PEX10 and observed that its ex- nuclear genes, synthesized on cytoplasmic ribosomes, pression rescues peroxisomal matrix-protein import in and posttranslationally imported into peroxisomes (La- PBD patients’ fibroblasts from complementation group zarow and Fujiki 1985). The import process, which is 7 (CG7). In addition, we detected mutations on both conserved from yeast to humans (Gould et al. 1990), is copies of PEX10 in two unrelated CG7 patients. A Zell- mediated by cis-acting peroxisome-targeting signals weger syndrome patient, PBD100, was homozygous for (PTS). For peroxisomal matrix proteins, import is most a splice donor–site mutation that results in exon skip- commonly specified by the type-1 PTS (PTS1), which ping and loss of 407 bp from the PEX10 open reading consists of a C-terminal tripeptide of the sequence Ser- frame. A more mildly affected neonatal adrenoleuko- Lys-LeuCOOH (SKLCOOH) or a conservative variant (Gould dystrophy patient was a compound heterozygote for a et al. 1989; Subramani 1993). Less commonly used is missense mutation in the PEX10 zinc-binding domain, the PTS2, a 9-amino-acid signal located near the amino H290Q, and for a nonsense mutation, R125ter. Al- terminus of proteins with a consensus sequence RLX5H/ though all three mutations attenuate PEX10 activity, the QL (Subramani 1993; Swinkels et al. 1991). Although two alleles detected in the mildly affected patient, peroxisomal membrane proteins also contain cis-acting PBD052, encode partially functional PEX10 proteins. sequence elements that target them to peroxisomes (Dyer PEX10-deficient PBD100 cells contain many peroxi- et al. 1996), a consensus sequence has yet to be somes and import peroxisomal membrane proteins but established. do not import peroxisomal matrix proteins, indicating The identification of trans-acting components of the that loss of PEX10 has its most pronounced effect on peroxisomal protein import machinery has relied heavily peroxisomal matrix-protein import. on genetic screens and selections in yeast. These studies have identified at least 16 different genes (PEX) and gene products (peroxins) that are required for peroxisome bi- ogenesis and normal matrix-protein import (Distel et al. 1996; Albertini et al. 1997; Eitzen et al. 1997) (S.J.G., unpublished results). Information derived from studying yeast pex mutants, which have a defect in the import of Received January 21, 1998; accepted for publication June 8, 1998; one or more classes of peroxisome matrix proteins, has electronically published July 10, 1998. shaped our understanding of peroxisome protein import. Address for correspondence and reprints: Dr. Stephen J. Gould, De- Import is thought to begin with two predominantly cy- partment of Biological Chemistry, The Johns Hopkins University toplasmic receptors, PEX5 and PEX7, that recognize School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. newly synthesized proteins that contain a PTS (Mc- E-mail: [email protected] ᭧ 1998 by The American Society of Human Genetics. All rights reserved. Collum et al. 1993; Marzioch et al. 1994; Dodt et al. 0002-9297/98/6302-0010$02.00 1995; Dodt and Gould 1996; Elgersma et al. 1996; 347 348 Am. J. Hum. Genet. 63:347–359, 1998 Gould et al. 1996; Braverman et al. 1997). PEX5 binds with 10% fetal bovine serum and penicillin/streptomycin PTS1-containing proteins, whereas PEX7 binds PTS2- (Life Technologies-BRL), as described elsewhere (Slaw- containing proteins, and together these receptors direct ecki et al. 1995). All PBD cell lines are referred to by newly synthesized matrix proteins to the peroxisome. patient number and were obtained from Ann Moser The receptor-bound proteins are recognized by (Kennedy Krieger Institute, Baltimore). All bacterial ma- membrane-anchored docking factors on the cytoplasmic nipulations were performed with Escherichia coli strain face of peroxisome membranes and subsequently are DH10B. translocated into the peroxisome matrix, presumably through a membrane pore, although there is no evidence Cloning of Human PEX10 yet for such a structure. PCR primers PEX10.1 (5 -AGG TTG AGC TGC TCT The peroxisome-biogenesis disorders (PBDs) are a ge- CAG ATG TGG-3 ) and PEX10.2 (5 -GAT GGG TCC netically heterogeneous group of lethal human diseases ACC GGA TGA TGC-3 ) were designed from the se- that are caused by an inability to import peroxisomal quence of EST H83562 and were used to amplify a 103- proteins (Lazarow and Moser 1995), the same pheno- bp fragment of the putative PEX10 cDNA from a human type displayed by yeast pex mutants. The PBDs include muscle cDNA library (Clontech). This fragment, which Zellweger syndrome (ZS), neonatal adrenoleukodystro- encodes the conserved TLGEEYV motif, was used to phy (NALD), infantile Refsum disease (IRD), and rhi- probe a lZAP human fetal brain cDNA library (Stra- zomelic chondrodysplasia punctata (RCDP) (Lazarow tagene) at high stringency (Sambrook et al. 1989). Of and Moser 1995), and cell fusion studies have divided the 16 independent library clones that hybridized to the the PBDs into 10 complementation groups (CGs) (Moser PEX10 probe, 6 were sequenced from both ends of the et al. 1995). ZS, NALD, and IRD are characterized by cDNA. The entire cDNA insert of one of the clones, 28- similar symptoms, with ZS representing the most severe 2.3, was sequenced and was found to contain an open form and with NALD and IRD representing progres- reading frame (ORF) (GenBank accession number sively less severe phenotypic variants (Lazarow and AF060502) similar in size to those of the yeast PEX10 Moser 1995). Seven of the known CGs contain both genes. mildly and severely affected patients, suggesting that the different phenotypes can result from mutations in the Plasmids same gene (Moser et al. 1995). The phenotype of clas- sical RCDP patients, which all belong to CG11, is some- To generate the PEX10 cDNA expression plasmid what distinct from the phenotypic spectrum of ZS, (cDNA-PEX10) a 1.1-kb NgoMI fragment, containing NALD, and IRD and is caused by defective import of the entire PEX10 ORF, was excised from cDNA clone PTS2-containing proteins (Braverman et al. 1997; Mot- 28-2.3, and the Klenow fragment of DNA polymerase ley et al. 1997; Purdue et al. 1997). (Boehringer Mannheim) was used to make the overhangs Given the conserved nature of the peroxisomal pro- blunt. This fragment was ligated with pcDNA3 (Invi- tein-import process (Gould et al. 1990), it is not sur- trogen) that had been digested with EcoRI and that had prising that the PBDs are caused by mutations in human been made blunt by means of the Klenow fragment. The orthologues of yeast PEX genes. This was first dem- PEX10myc cDNA was created by amplification of the onstrated by the identification of human PEX5, the gene PEX10 ORF (lacking its stop codon), with the primers mutated in PBD CG2 (Dodt et al. 1995; Wiemer et al. PEX10myc5 (5-CCC GGT ACC ATG GCC CCG GCC 1995), and has been confirmed by the identification of GCC GCC AG-3) and PEX10myc3 (5-CCC GGA TCC human PEX1, PEX6, PEX7, and PEX12 as the genes GCG GTA GTG CCG AAG GTA G-3). The resulting defective in CG1 (Portsteffen et al. 1997; Reuber et al. fragment was cleaved with Asp718 and BamHI and was 1997), CG4 (Fukuda et al. 1996; Yahraus et al. 1996), cloned in-frame with the myc-epitope-tag–encoding se- CG11 (Braverman et al. 1997; Motley et al. 1997; Pur- quence in pcDNA3myc (Yahraus et al. 1996). The due et al. 1997), and CG3 (Chang et al. 1997; Okumoto pcDNA3-PEX10myc clone was sequenced, ensuring and Fujiki 1997) of the PBDs, respectively. We report that no errors were incorporated during PCR amplifi- here the cloning of the human orthologue of yeast cation. PEX10 cDNA was synthesized from PBD052 PEX10 and demonstrate that it is the gene defective in and PBD100 RNA by reverse transcription (RT)–PCR CG7 of the PBDs. and was cloned into PCR 2.1 (Invitrogen), as described in the “Mutation Analysis” section below. The three Material and Methods mutant cDNAs identified in these patients were excised by EcoRI restriction-endonuclease digestion and were Cell Lines and Strains inserted into the EcoRI site of pcDNA3. The resulting Skin fibroblast cell lines were cultured in Dulbecco’s plasmids, pcDNA3-PEX10/H290Q, pcDNA3-PEX10/ modified Eagle’s medium (high glucose) supplemented R125ter, and pcDNA3-PEX10/PBD100, were used in Warren et al.: PEX10 Is Defective in CG7 of the PBDs 349 the relative rescue experiments.